Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Alan J. Biel

Abstract

The common tetrapyrrole pathway in the photosynthetic bacterium, Rhodobacter capsulatus, is responsible for the synthesis of bacteriochlorophyll, heme, vitamin B-12, and siroheme. The common portion of this pathway is regulated up to 100-fold by changes in oxygen tension. This regulation is accomplished by controlling the intracellular level of porphobilinogen. One way in which porphobilinogen levels could be controlled is by regulating the synthesis of porphobilinogen. Porphobilinogen synthase, encoded by the hemB gene, is the second enzyme in the common tetrapyrrole pathway that catalyzes the dimerization of 5-aminolevulinic acid to form the monopyrrole porphobilinogen. In order to further investigate the mechanism by which oxygen regulates porphobilinogen levels, the R. capsulatus hemB gene was cloned and sequenced. The R. capsulatus hemB gene was cloned by complementation of an Escherichia coli hemB mutant. Sequence analysis of the R. capsulatus hemB gene revealed that the putative porphobilinogen synthase has a metal-binding domain that more closely resembles that found in plant porphobilinogen synthases. The locations of the hemB and hemA genes on the R. capsulatus chromosome indicate that these genes do not form an operon. Oxygen mediated transcriptional regulation of the R. capsulatus hemB gene was measured by dot blot analysis of mRNA from cells grown under 3% and 23% oxygen and by hemB-cat transcriptional fusion studies. Both of these methods reveal that the synthesis of porphobilinogen synthase does not change with shifts in oxygen tension. Oxygen tension also does not appear to regulate enzyme activity since the specific activity does not change significantly with changes in oxygen tension. Overexpression of the R. capsulatus hemB, in the presence of exogenous aminolevulinate, in R. capsulatus can overcome the normal oxygen mediated regulatory mechanisms. While the mechanism of how oxygen modulates carbon flow down the common tetrapyrrole pathway remains elusive, it is clear that the level of porphobilinogen plays a crucial part in oxygen regulation. The possibility still exists that oxygen regulates the degradation of porphobilinogen.

Pages

86

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